Methods and compositions for protein production in tobacco plants with reduced nicotine

ABSTRACT

The present application describes isolated nucleic acids that contain a Nic gene product responsive element, and the use thereof in methods of producing transgenic tobacco plants having reduced levels of nicotine and/or TSNA therein, as well as other plants or host cells that contain altered levels of a protein of interest therein due to inclusion of a cis-acting decoy element therein.

RELATED APPLICATIONS

This application is a continuation application of, and claims priorityto, U.S. application Ser. No. 10/985,401, filed Nov. 10, 2004, whichstatus is pending, which is a continuation application of U.S.application Ser. No. 09/941,042, filed Aug. 28, 2001 and issued as U.S.Pat. No. 6,911,541 on Jun. 28, 2005, and which claims the benefit ofU.S. Provisional Application No. 60/229,198, filed Aug. 30, 2000. Theentire contents of each of these applications is incorporated byreference herein.

FIELD OF THE INVENTION

The present invention describes a process for the production oftransgenic plants such as transgenic tobacco plants with altered proteincontent therein, leading to altered phenotypes such as reduced nicotinelevels, along with transgenic plants so produced and seed for suchplants.

BACKGROUND OF THE INVENTION

The production of tobacco with decreased levels of nicotine is ofinterest, given concerns regarding the addictive nature of nicotine.Additionally, tobacco plants with extremely low levels of nicotineproduction, or no nicotine production, are attractive as recipients fortransgenes expressing commercially valuable products such aspharmaceuticals, cosmetic components, or food additives. Variousprocesses have been designed for the removal of nicotine from tobacco.However, most of these processes remove other ingredients from tobaccoin addition to nicotine, thereby adversely affecting the tobacco.Classical crop breeding techniques have produced tobacco plants withlower levels of nicotine (approximately 8%) than that found in wild-typetobacco plants. Tobacco plants and tobacco having even furtherreductions in nicotine content are desirable.

Nicotine is formed primarily in the roots of the tobacco plant and issubsequently transported to the leaves, where it is stored (Tso,Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden,Hutchinson & Ross, Stroudsburg, Pa. (1972)). Nicotine is produced by thecondensation of two precursors, nicotinic acid and N-methylpyrolinium,that arise from two separate biosynthetic pathways (see FIG. 1)(Bush andSaunders (1977) Proc. Am. Chem. Soc. Symp., New Orleans, pp. 389-425;Hashimoto and Yamada (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol.45, 257-285; Waller and Dermer (1981) In: The Biochemistry of Plants: AComprehensive Treatise, P. K. Stumpf and E. E. Conn, eds. AcademiaPress, pp. 317-395). The pyridine nucleotide cycle synthesize nicotinicacid (Wagner et al. (1986) Planta 167, 226-232; Wagner and Wagner (1985)Planta 165, 532-537), whereas N-methylpyrrolinium cations aresynthesized from ornithine or arginine via putrescence (Leete (1980) In:Encyclopedia of Plant Physiology, Secondary Plant Products, Vol. 8, E.A. Bell and B. V. Charlwood, eds, Springer-Verlag, pp. 65-91; Tiburcioand Galston (1986) Phytochemistry, 25, 107-110). Reciprocal graftingexperiments have demonstrated that nicotine is synthesized in roots andtransported through the xylem to leaves and other plant organs (Dawson(1941) Science, 94, 396-397).

Two regulatory loci (Nic1 and Nic2) regulate nicotine production. Legget al. ((1969) J. Hered., 60, 213-217) incorporated genes from lowalkaloid content Cuban cigar cultivars into Burley 21 cultivars. Theseinvestigators showed that the low alkaloid lines differed from standardcultivars at two loci, Nic1 (formerly identified as A) and Nic2(formerly identified as B). These two loci are unlinked and the geneaction is semi-dominant and primarily additive (Legg et al. (1969) J.Hered., 60, 213-217). Collins et al. ((1974) Crop Sci., 14, 77-80)prepared doubled haploid tobacco breeding lines of these four alkaloidgenotypes. The genotype of standard cultivars is Nic1/Nic1 Nic2/Nic2 andthat of low nicotine lines is nic1/nic1 nic2/nic2. Nic1/Nic1 nic2/nic2is a high intermediate and nic1/nic1 Nic2/Nic2 is a low intermediate(Legg and Collins (1971) Can. J. Genet. Cytol. 13, 287-291). These linesare similar in days-to-flower, number of leaves, leaf size, and plantheight. Enzyme analyses of roots of single and double Nic mutants showthat the activities of two enzymes, quinolinate phosphoriboxyltransferase (QPTase) and putrescence methyl transferase (PMTase), aredirectly proportional to levels of nicotine biosynthesis (Saunders andBush (1979) Plant Physiol 64:236). Both Nic1 and Nic2 affect PMTase andQPTase activities in roots, and thus, regulate nicotine synthesis (Leete(1983) In: Alkaloids: Chemical and Biological Perspectives, S. W.Pelletier, ed. John Wiley & Sons, pp. 85-152).

Hibi et al. ((1994) Plant Cell, 6, 723-735) isolated the cDNA encodingPMTase, PMT, and showed that PMT transcript levels are regulated by Nic1and Nic2. The QPTase cDNA and genomic clones (NtQPT1) have also beenisolated and the transcript levels of NtQPT1 are also regulated by Nic1and Nic2 (Song, W., Mendu, N., and Conkling, M. A. (1999) Plant Cell, inpreparation). Thus, it appears that the Nic genes regulate nicotinecontent by regulating the transcript levels of genes encoding the tworate-limiting enzymes, PMTase and QPTase. Further, Nic1 and Nic2 havebeen shown to be positive regulators of NtQPT1 transcription and thatpromoter sequences upstream of the transcription initiation site containthe cis-acting sequences necessary for Nic gene product activation ofNtQPT1 transcription. Because expression of QPTase and PMTase arecoordinately-regulated by the Nic gene products, it likely that the Nicgene products also directly regulate transcription of the PMT gene.

One approach for reducing the level of a biological product, such asnicotine, is to reduce the amount of a required enzyme (i.e. QPTase andPMTase) in the biosynthetic pathway leading to that product. Where theaffected enzyme naturally occurs in a rate-limiting amount (relative tothe other enzymes required in the pathway), any reduction in thatenzyme's abundance will decrease the production of the end product. Ifthe amount of the enzyme is not normally rate-limiting, its presence ina cell must be reduced to rate-limiting levels in order to diminish thepathway's output. Conversely, if the naturally-occurring amount ofenzyme is rate limiting, then any increase in the enzyme's activity willresult in an increase in the biosynthetic pathway's end product. Themodification of nicotine levels in tobacco plants by antisenseregulation of putrescence methyl transferase (PMTase) expression isproposed in U.S. Pat. Nos. 5,369,023 and 5,260,205 to Nakatani andMalik. PCT application WO 94/28142 to Wahad and Malik describes DNAencoding PMT and the use of sense and antisense PMT constructs.Additionally, PCT Application WO98/56923 to Conkling et al. describesDNA encoding a plant quinolate phosphoribosyl transferase (QPRTase)enzyme, constructs comprising such DNA, and methods of altering QPRTaseexpression to increase or decrease nicotine production in plants.Despite previous efforts and successes, there remains a need for newapproaches to reduce the production of gene products in plants ( e.g.,nicotine).

SUMMARY OF THE INVENTION

A first aspect of the present invention is an isolated nucleic acidmolecule (e.g., a plasmid) comprising, consisting essentially of, orconsisting of a cis-acting regulatory element, and the use of such anisolated nucleic acid for the production of a transgenic plant or hostcell having altered levels (e.g., increased or decreased levels) of aprotein of interest therein. A particular example is a Nic gene productresponsive element (e.g., a DNA sequence that binds to a Nic geneproduct) such as (a) isolated nucleic acids having a sequence accordingto SEQ ID NO: 1 or a fragment thereof consisting essentially of orconsisting of, desirably, at least 20-455 consecutive nucleotides,preferably, at least 30-400 consecutive nucleotides, more preferably,50-350 consecutive nucleotides, and, most preferably, 100-300 or 200-400consecutive nucleotides; and (b) isolated nucleic acids that hybridizeto the complement of SEQ ID NO:1 and bind or are otherwise responsive toa Nic gene product (i.e., increase or decrease transcription of anoperatively associated gene and hence increase or decrease the level ofthe encoded protein of interest in the host cells).

The Nic gene product responsive element can also be obtained from thesequence disclosed in U.S. Pat. No. 5,459,252, herein expresslyincorporated by reference in its entirety. In some embodiments, the Nicgene product responsive element resides between −1000 and −600 or −700bp of the NtQPT1 promoter, the sequence of which is disclosed in U.S.Pat. No. 5,459,252. Accordingly, some embodiments involve a 300-400nucleotide long fragment of the NtQPT1 promoter that corresponds to thesequence of the NtQPT1 promoter between −1000 and −600 or −700, asdisclosed in U.S. Pat. No. 5,459,252.

A second aspect of the present invention is a recombinant nucleic acidconstruct comprising containing a cis-acting regulatory element such asa Nic gene product responsive element as described above, along with theuse of such a recombinant nucleic acid for the production of atransgenic plant or host cell as described herein. The construct may bea vector, such as a ballistic nucleic acid transfer particle or anAgrobacterium vector. Plant cells containing such constructs, andpreferably multiple copies thereof, are also an aspect of the invention.

A further aspect of the present invention is a method of making atransgenic tobacco plant having reduced nicotine content and/or tobaccospecific nitrosamines (TSNA)s. The method comprises introducing anexogenous nucleic acid construct comprising a Nic gene productresponsive element as described above into said at least one tobaccoplant cell to produce at least one transformed tobacco plant cell. Theat least one transformed tobacco plant cell contains the exogenousnucleic acid in an amount or copy number sufficient to reduce thenicotine and/or TSNA level of a tobacco plant regenerated from that cellor cells as compared to the nicotine and/or TSNA level that would bepresent in the absence of the exogenous nucleic acid. The method mayfurther include generating a tobacco plant from the transformed plantcells, and (optionally) collecting tobacco leaves, stems, or seed fromthe tobacco plant. Thus, tobacco plants, including the leaves, stems,and seeds, generated from said method are also aspects of the presentinvention.

A further aspect of the present invention is a tobacco plant havingreduced levels of nicotine and/or TSNAs therein, the plant comprisingcells containing an exogenous nucleic acid, which exogenous nucleic acidcomprises a Nic gene product responsive element as described above. Theexogenous nucleic acid is contained in the cells in a copy numbersufficient to reduce the nicotine level of that tobacco plant ascompared to the nicotine level that would be present in that plant inthe absence of the exogenous nucleic acid. Again, the leaves, stems, andseeds of such plant are also aspects of the present invention.

Tobacco products including, but not limited to, smoking materials (e.g.,cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, lozengesthat are prepared from said transgenic tobacco plants are alsoembodiments of the invention. Preferably these tobacco products aremanufactured from harvested tobacco leaves and stems that have been cut,dried, cured, and/or fermented according to conventional techniques intobacco preparation. However, modified techniques in curing and tobaccoprocessing can also be implemented to further lower the levels of TSNAs.In some embodiments, the tobacco that is made substantially free ofnicotine and/or TSNAs is prepared from a variety of Burley tobacco(e.g., Burley 21), Oriental tobacco, or Flue-cured tobacco. It should beunderstood, however, that most tobacco varieties can be made to benicotine and/or TSNA free using the embodiments described herein.

Additional embodiments include tobacco products that have been carefullyblended so that desired levels of nicotine and/or TSNAs are obtained.For example, tobacco having a reduced level of nicotine and/or TSNAs,prepared as described above, can be blended with conventional tobacco soas to obtain virtually any amount of nicotine and/or TSNAs. Further, twoor more varieties of tobacco having a reduced level of nicotine and/orTSNAs can be blended so as to achieve a desired amount of nicotineand/or TSNAs. In this manner, differences in variety, flavor, as well asamounts of nicotine and/or TSNAs can be incrementally adjusted. Theseblended tobacco products can be incorporated into tobacco use cessationkits and programs designed to reduce or eliminate nicotine dependenceand carcinogenic potential. Such kits and programs are also embodimentsof the invention.

More embodiments of the invention concern methods to reduce thecarcinogenic potential of tobacco products, including cigarettes,cigars, chewing tobacco, snuff and tobacco-containing gum and lozenges.Some methods, for example involve the preparation of tobacco having areduced amount of nicotine and/or TSNAs and the manufacture of tobaccoproducts containing said tobacco. Accordingly, the transgenic tobaccoplants, described above, are harvested, cured, and processed intotobacco products. These tobacco products have a reduced carcinogenicpotential because they are prepared from tobacco that has a reducedamount of nicotine and/or TSNAs.

Yet another aspect of the invention concerns the reduction of the amountof TSNAs and metabolites thereof in humans who smoke, consume orotherwise ingest tobacco. This method is practiced by providing atobacco product having a reduced amount of TSNAs, as described above, tosaid humans, thereby lowering the carcinogenic potential of such productin said humans.

More generally, the present invention provides a method of making aplant having increased or reduced content of a protein of interesttherein, wherein the protein of interest is regulated by a cis-actingelement selected from the group consisting of (i) a cis-actingactivating element that binds an activator compound, which activatorcompound increases expression of said protein of interest in said plant,and (ii), a cis-acting repressor element that binds a repressorcompound, which repressor compound decreases expression of said proteinof interest in said plant. The method comprises introducing an exogenousnucleic acid construct comprising said cis-acting element into at leastone plant cell to produce at least one transformed plant cell, with theat least one transformed plant cell containing the exogenous nucleicacid in a copy number sufficient to increase or reduce the level of saidprotein of interest in a plant regenerated from said cells as comparedto the amount of said protein of interest that would be present in theabsence of said exogenous nucleic acid.

The present invention thus generally provides a plant (and partsthereof) having increased or reduced levels of a protein of interesttherein, the plant comprising cells containing an exogenous nucleicacid, which exogenous nucleic acid comprises a cis-acting elementselected from the group consisting of (i) a cis-acting activatingelement that binds an activator compound, which activator compoundincreases expression of said protein of interest in said plant, and(ii), a cis-acting repressor element that binds a repressor compound,which repressor compound decreases expression of said protein ofinterest in said plant; the cells containing the exogenous nucleic acidin a copy number sufficient to increase or reduce the level of theprotein of interest in the plant as compared to the amount of theprotein of interest that would be present in the absence of theexogenous nucleic acid.

Thus the present invention provides a general method of decreasingexpression of a protein of interest in a (prokaryotic or eukaryotic)host cell, wherein transcription of the protein of interest is enhancedby a cis-acting activating element that binds an activator compound,which activator compound increases expression of the protein of interestin the host cell. The method comprises the steps of: (a) providing adecoy recombinant nucleic acid construct comprising the cis-actingactivating element; and (b) introducing the decoy construct into thehost cell in an amount sufficient to bind the activator compound andreduce expression of the protein of interest.

Further, the present invention provides a general method of increasingexpression of a protein of interest in a host cell, whereintranscription of the protein of interest is reduced by a cis-actingrepressor element that binds a repressor compound, which repressorcompound reduces expression of said protein of interest in said hostcell. The method comprises the steps of: (a) providing a decoyrecombinant nucleic acid construct comprising said cis-acting activatingelement; and (b) introducing said decoy construct into said host cell inan amount sufficient to bind said repressor compound and increaseexpression of said protein of interest.

The foregoing and other aspects of the present invention are explainedin greater detail in the drawings herein and the specification set forthbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the biosynthetic pathway leading to nicotinebiosynthesis. Enzyme activities known to be regulated by Nic1 and Nic2are QPTase (quinolinate phosphoribosyl transferase) and PMTase(putrescence methyl-transferase). QPTase and PMTase are therate-limiting enzymatic steps in nicotine biosynthesis and thus,nicotine levels are directly proportional to the QPTase and PMTaseactivities.

FIG. 2 shows a diagrammatic representation of the NtQPT1 gene and theNtQPT1 promoter-uidA chimeras. The site of transcription initiation isindicated (+1) and the arrow indicates the NtQPT1 transcript. Ten exonsare presented as crosshatched bars. The deletion series of the promoteris also shown as solid bars truncated from the 5′ end of the promoter.Sizes of promoter fragments fused to the uidA gene, which encodesβ-glucuronidase (GUS) are indicated (i.e. Δ2.0, Δ1.4, etc.) in kilobasepairs (kb). Chimeric NtQPT1 promoter-uidA fusions were cloned intopBI101.

FIG. 3 shows β-glucuronidase (GUS) activity in roots, leaves, and stemsof transgenic tobacco plants carrying the CaMV 35S promoter (CaMV 35S),the promoterless GUS (pBI101), and 5′ nested deletions of the TobRD2(gene encoding NtQPT1) promoter fused to GUS. Sizes of promoterfragments fused to the uidA gene are indicated (i.e. Δ2.0, Δ1.4, etc.)in kilobase pairs (kb). For each construct at least 20 independenttransformants were assayed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The term “plants” as used herein refers to vascular plants. Exemplaryplants include, but are not limited, to corn (Zea mays), canola(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice(Oryza sativa), rape (Brassica napus), rye (Secale cereale), sorghum(Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), apple (Malus pumila),blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape(Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach(Prunus persica), plum (Prunus domestica), pear (Pyrus communis),watermelon (Citrullus vulgaris), duckweed (Lemna), oats, barley,vegetables, ornamentals, conifers, and turfgrasses (e.g., forornamental, recreational or forage purposes). Vegetables includeSolanaceous species (e.g., tomatoes; Lycopersicon esculentum), lettuce(e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassicaoleracea), celery (apium graveolens), eggplant (Solanum melongena),asparagus (Asparagus officinalis), ochra (Abelmoschus esculentus), greenbeans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas(Lathyrus spp.), members of the genus Cucurbita such as Hubbard squash(C. Hubbard), Butternut squash (C. moschata), Zucchini (C. pepo),Crookneck squash (C. crookneck), C. argyrosperma, C. argyrosperma sspsororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana,and C. martinezii, and members of the genus Cucumis such as cucumber(Cucumis sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamental plants include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherima), and chrysanthemum. Conifers, which may beemployed in practicing the present invention, include, for example,pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Turfgrass include but are not limited to zoysiagrasses, bentgrasses,fescue grasses, bluegrasses, St. Augustinegrasses, bermudagrasses,buffalograsses, ryegrasses, and orchardgrasses. Also included are plantsthat serve primarily as laboratory models, e.g., Arabidopsis. Preferredplants for use in the present methods include (but are not limited to)legumes, solanaceous species (e.g., tomatoes), leafy vegetables such aslettuce and cabbage, turfgrasses, and crop plants (e.g., tobacco, wheat,sorghum, barley, rye, rice, corn, cotton, cassava, and the like), andlaboratory plants (e.g., Arabidopsis). While any plant may be used tocarry out the present invention, tobacco plants are particularlypreferred.

Plant parts that can be collected from the plants of the presentinvention (e.g., cut or harvested) include, for example, fruits,flowers, seed, roots, tubers, leaves, stems, bark, wood, etc. Note thatwhen reference is made to a particular protein being increased orreduced in a plant, the amount of that protein may be altered throughoutthe plant, or only in a particular part of the plant.

In overview, in an illustrative embodiment of the invention, nicotine isproduced in tobacco plants by the condensation of nicotinic acid andN-methylpyrrolinium cation. The biosynthetic pathway resulting innicotine production is illustrated in FIG. 1. Two regulatory loci (Nic1and Nic2) act as co-dominant regulators of nicotine production. Enzymeanalyses of roots of single and double Nic mutants show that theactivities of two enzymes, quinolate phosphoribosyl transferase (QPTase)and putrescence methyl transferase (PMTase), are directly proportionalto levels of nicotine biosynthesis. A comparison of enzyme activity intobacco tissues (root and callus) with different capacities for nicotinesynthesis shows that QPTase and PMTase activity are strictly correlatedwith nicotine content (Wagner and Wagner, Planta 165:532 (1985)).Saunders and Bush (Plant Physiol 64:236 (1979) showed that the level ofQPTase in the roots of low nicotine mutants is proportional to thelevels of nicotine in the leaves.

The present invention is, in one preferred embodiment, based upon anisolated nucleic acid (e.g., SEQ ID NO:1 or a fragment thereofconsisting of, desirably, at least 20-450 consecutive nucleotides,preferably, at least 30-400 consecutive nucleotides, more preferably,50-350 consecutive nucleotides, and, most preferably, 100-300 or 200-400consecutive nucleotides) that is or contains at least one cis-actingregulatory element, which exists upstream of the plant quinolatephosphoribosyl transferase (QPTase) and putrescence methyl transferase(PMTase) coding sequences. Another example is the Nic gene productresponsive element obtained from the sequence disclosed in U.S. Pat. No.5,459,252, herein expressly incorporated by reference in its entirety.In some embodiments, the Nic gene product responsive element residesbetween −1000 and −600 or −700 bp of the NtQPT1 promoter. Accordingly,some embodiments involve a 300-400 nucleotide long fragment of theNtQPT1 promoter that corresponds to the sequence of the NtQPT1 promoterbetween −1000 and −600 or −700, as disclosed in U.S. Pat. No. 5,459,252.

Thus, in some embodiments, the embodied nucleic acids have a structurethat promotes an interaction with one or more transcription factors(e.g., Nic1 and Nic2), which are involved in initiating transcription ofQPTase and/or PMTase. Accordingly, said nucleic acids are said to be orcontain at least one transcription factor (e.g., Nic1 and Nic2) bindingsequences, which are also referred to as “cis-acting regulatoryelements.” By introducing multiple copies of these cis-acting regulatoryelements (e.g., sequences that interact with Nic1 and/or Nic2) into aplant cell, the ability of the transcription factor to initiatetranscription of the targeted gene (e.g., QPTase and/or PMTase genes)can be reduced or squelched.

As QPTase and PMTase activities are strictly correlated with nicotinecontent, construction of transgenic tobacco plants in which QPTase orPMTase levels are lowered in the plant roots (compared to levels inwild-type plants), as described above, result in plants having reducedlevels of nicotine. Without wishing to be bound by any particulartheory, it is contemplated that the creation of tobacco plants, tobacco,and tobacco products that have a reduced amount of nicotine will alsohave a reduced amount of TSNA. That is, by removing nicotine fromtobacco plants, tobacco, and tobacco products, one effectively removesthe alkaloid substrate for TSNA formation. It was found that thereduction of nicotine in tobacco was directly related to the reductionof TSNAs. Unexpectedly, the methods described herein not only producetobacco with a reduced addictive potential but, concomitantly, produce atobacco that has a lower carcinogenic potential.

It should be emphasized that the phrase “a reduced amount” is intendedto refer to an amount of nicotine and or TSNA in a transgenic tobaccoplant, tobacco, or a tobacco product that is less than what would befound in a tobacco plant, tobacco, or a tobacco product from the samevariety of tobacco processed in the same manner, which was not madetransgenic for reduced nicotine and/or TSNA. Thus, in some contexts,wild-type tobacco of the same variety that has been processed in thesame manner is used as a control by which to measure whether a reductionin nicotine and/or TSNA has been obtained by the inventive methodsdescribed herein.

Wild type tobacco varies significantly in the amount of TSNAs andnicotine depending on the variety and the manner it is grown, harvested,and cured. For example, a Burley tobacco leaf has 30,000 parts permillion (ppm) nicotine and 8,000 parts per billion (ppb) TSNA; aFlue-Cured Burley leaf has 20,000 ppm nicotine and 300 ppb TSNA; and anOriental cured leaf has 10,000 ppm nicotine and 100 ppb TSNA. A tobaccoplant or portion thereof having a reduced amount of nicotine and/orTSNA, according to the invention, can have no detectable nicotine and/orTSNA, or may contain some detectable amounts of one or more TSNA and/ornicotine so long as the amount of nicotine and/or TSNA is less than thatfound in a control plant of the same variety. That is, a Burley tobaccoleaf embodiment of the invention having a reduced amount of nicotine canhave between 0 and 30,000 ppm nicotine and 0 and 8,000 ppb TSNAdesirably between 0 and 20,000 ppm nicotine and 0 and 6,000 ppb TSNAmore desirably between 0 and 10,000 ppm nicotine and 0 and 5,000 ppbTSNA preferably between 0 and 5,000 ppm nicotine and 0 and 4,000 ppbTSNA more preferably between 0 and 2,500 ppm nicotine and 0 and 2,000ppb TSNA and most preferably between 0 and 1,000 ppm nicotine and 0 and1,000 ppb TSNA. Embodiments of Burley leaf prepared by the methodsdescribed herein can also have between 0 and 1000 ppm nicotine and 0 and500 ppb TSNA and some embodiments of Burley leaf prepared by the methodsdescribed herein have virtually no detectable amount of nicotine orTSNA.

Similarly, a Flue-cured tobacco leaf embodiment of the invention havinga reduced amount of nicotine can have between 0 and 20,000 ppm nicotineand 0 and 300 ppb TSNA desirably between 0 and 15,000 ppm nicotine and 0and 250 ppb TSNA more desirably between 0 and 10,000 ppm nicotine and 0and 200 ppb TSNA preferably between 0 and 5,000 ppm nicotine and 0 and150 ppb TSNA more preferably between 0 and 2,500 ppm nicotine and 0 and100 ppb TSNA and most preferably between 0 and 1,000 ppm nicotine and 0and 50 ppb TSNA. Embodiments of flue-cured tobacco prepared by themethods described herein can also have between 0 and 500 ppm nicotineand 0 and 25 ppb TSNA and some embodiments of flue-cured tobaccoprepared by the methods described herein have virtually no detectableamount of nicotine or TSNA.

Further, an Oriental cured tobacco embodiment of the invention having areduced amount of nicotine can have between 0 and 10,000 ppm nicotineand 0 and 100 ppb TSNA desirably between 0 and 7,000 ppm nicotine and 0and 75 ppb TSNA more desirably between 0 and 5,000 ppm nicotine and 0and 50 ppb TSNA preferably between 0 and 3,000 ppm nicotine and 0 and 25ppb TSNA more preferably between 0 and 1,500 ppm nicotine and 0 and 10ppb TSNA and most preferably between 0 and 500 ppm nicotine and no TSNA.Embodiments of Oriental cured tobacco prepared by the methods describedherein can also have between 0 and 250 ppm nicotine and no TSNA and someembodiments of Oriental cured tobacco prepared by the methods describedherein have virtually no detectable amount of nicotine or TSNA.

The present invention provides methods and nucleic acid constructs forproducing such transgenic plants, as well as such transgenic plants.Such methods include the development of transgenic cassettes that willreduce (or eliminate) nicotine biosynthesis. Tobacco plants aretransformed with an excess number of DNA sequences (cis-acting elements)from the promoters of genes encoding, but not limited to, QPTase andPMTase that are regulated in nicotine biosynthesis. These cis-actingelements are preferably integrated into the plant genome so as to allowfor transfer to successive generations. Typically, the Nic1 and Nic2DNA-binding proteins that interact with these cis-acting DNA sequencesare expressed at relatively low levels in the cell, thus the excess oftransgenic cis-acting elements will compete with the endogenous elementsassociated with the genes encoding, but not limited to, QPTase andPMTase for available Nic1 and Nic2. Accordingly, these cis-acting DNAsequences (and those of other cis-acting elements) are referred toherein as “decoys” or “molecular decoys”. The competition decreasesoccupancy of trans-acting DNA-binding proteins on their cognatecis-acting elements, thereby down-regulating the synthesis of nicotinebiosynthesis enzymes.

The present invention also provides DNA molecules of cis-acting elementsof QPTase or PMTase, and vectors comprising those DNA molecules, as wellas transgenic plant cells and plants transformed with those DNAmolecules and vectors. Transgenic tobacco cells and plants of thisinvention are characterized by lower nicotine content than untransformedcontrol tobacco cells and plants.

Tobacco plants with low levels of nicotine production, or substantiallyno nicotine production, are attractive as recipients for transgenesexpressing commercially valuable products such as pharmaceuticals,cosmetic components, or food additives. Tobacco is attractive as arecipient plant for a transgene encoding a desirable product, as tobaccois easily genetically engineered and produces a very large biomass peracre; tobacco plants with reduced resources devoted to nicotineproduction accordingly will have more resources available for productionof transgene products. Methods of transforming tobacco with transgenesproducing desired products are known in the art; any suitable techniquemay be utilized with the low nicotine tobacco plants of the presentinvention.

Tobacco plants according to the present invention with reduced QPTaseand PMTase expression and reduced nicotine levels will be desirable inthe production of tobacco products having reduced nicotine and/or TSNAcontent. The tobacco plants described herein are suitable forconventional growing and harvesting techniques (e.g. topping or notopping, bagging the flowers or not bagging the flowers, cultivation inmanure rich soil or without manure) and the harvested leaves and stemsare suitable for use in any traditional tobacco product including, butnot limited to, pipe, cigar and cigarette tobacco, and chewing tobaccoin any form including leaf tobacco, shredded tobacco, or cut tobacco.

It is also contemplated that the low nicotine and/or TSNA tobaccodescribed herein can be processed and blended with conventional tobaccoso as to create a wide-range of tobacco products with varying amounts ofnicotine and/or nitrosamines. These blended tobacco products can be usedin tobacco product cessation programs so as to slowly move a consumerfrom a high nicotine and TSNA product to a low nicotine and TSNAproduct. For example, a smoker can begin the program smoking blendedcigarettes having 10 mg of nicotine and 1.5 mg of nitrosamine, graduallymove to smoking cigarettes with 7 mg of nicotine and 1 mg ofnitrosamine, followed by cigarettes having 5.0 mg nicotine and 0.5 mgnitrosamine, followed by cigarettes having 2.0 mg nicotine and 0.25 mgnitrosamine, followed by cigarettes having 1.0 mg nicotine and no TSNAuntil the consumer decides to smoke only the cigarettes having virtuallyno nicotine and nitrosamines or quitting smoking altogether.Accordingly, the blended cigarettes described herein provide the basisfor an approach to reduce the carcinogenic potential in a human in astep-wise fashion.

1. Nucleic Acids Encoding cis-Acting Elements Such as Nic Gene ProductResponsive Elements.

Any of a variety of cis-acting elements can be used in carrying out thepresent invention, depending upon the particular application of thepresent invention. Examples of cis-acting elements (and correspondingtranscription factors) that may be used, alone or in combination withone another, in practicing the present invention include, but are notlimited to, AS-1 and ASF-1 (see U.S. Pat. Nos. 4,990,607 and 5,223,419),the AATT repeat element and PABF (see U.S. Pat. Nos. 5,834,236 and6,191,258), a wounding-responsive cis-acting element from potato(Siebert et al., Plant Cell 1:961-8 (1989)), an embryo-specificcis-acting element from bean (Bustos et al, Plant Cell 1:839-853(1989)), a root-specific cis-acting element from the tobacco RB7promoter (U.S. Pat. No. 5,459,252 and Yamamoto et al., Plant Cell3:371-382 (1991)), a positive poly(dA-dT) regulatory element and bindingprotein and negative CCCAA repeat element and binding protein (Wang etal., Mol. Cell Biol. 12:3399-3406 (1992)), a root-tip regulatory elementfrom the tobacco phytochrome A1 promoter of tobacco (Adam et al., PlantMol. Biol. 29:983-993 (1995)), an anaerobiosis-responsive element fromthe maize glyceraldehyde-3-phosphate dehydrogenase 4 gene (Geffers etal., Plant Mol. Biol. 43:11-21 (2000)), and a seed-specific regulatoryregion from an Arabidopsis oleosin gene (see U.S. Pat. No. 5,792,922),all of which are hereby expressly incorporated by reference in theirentireties.

The status of the art is such that large databases list identifiedcis-acting regulatory regions (e.g., Plant Cis-acting Regulatoryelements, “PLACE”, with some 1,340 entries, seehttp://www.dna.affrc.gojp/hotdocs/PLACE/, and Plant Cis-actingRegulatory Elements “PlantCARE”, which lists some 159 plant promoter,see http://sphinx.rug.ac.be:8080/PlantCARE/. The listed cis-actingregulatory elements in these databases and the cis-acting regulatoryelements that are provided in Raumbauts et al., Nucleic acids Research27:295-296 (1999), and Higo et al., Nucleic acids Research 27:297-300(1999) can be used with embodiments of the invention. Accordingly, thedatabases and references above are hereby expressly incorporated byreference in their entireties. Additional examples of cis-actingregulatory regions, which can be used with embodiments of the inventioninclude: Lacombe E, Van Doorsselaere J, Boerjan W, Boudet A M,Grima-Pettenati J, Characterization of cis-elements required forvascular expression of the cinnamoyl CoA reductase gene and forprotein-DNA complex formation Plant J 23: 663-676 (2000); Tilly J J,Allen D W, Jack T The CArG boxes in the promoter of the Arabidopsisfloral organ identity gene APETALA3 mediate diverse regulatory effectsDevelopment 125: 1647-1657 (1998); Cordes S., Deikman J., Margossian L.J., Fischer R. L. Interaction of a developmentally regulated DNA-bindingfactor with sites flanking two different fruit-ripening genes fromtomato Plant Cell 1(10):1025-1034 (1989); Hagen G., Martin G., Li Y.,Guilfoyle T. Auxin-induced expression of the soybean GH3 promoter intransgenic tobacco plants” Plant Mol. Biol. 17:567-569 (1991); PastugliaM., Roby D., Dumas C., Cock J. M., Rapid induction by wounding andbacterial infection of an S gene family receptor-like kinase in Brassicaoleracea, Plant Cell 9:1-13 (1997); Grierson C, Du J S, Zabala M T,Beggs K, Smith C, Holdsworth M, Bevan M Separate cis sequences and transfactors direct metabolic and developmental regulation of a potato tuberstorage protein gene Plant J 5:815-826 (1994); MBSI, Petunia hybrida MYBbinding site involved in flavonoid biosynthetic gene regulation, Koes R.E., Spelt C. E., van Den Elzen P. J. M., Mol J. N. M. Cloning andmolecular characterization of the chalcone synthase multigene family ofPetunia hybrida, Gene 81:245-257 (1989); Inaba T., Nagano Y., SakakibaraT., Sasaki Y., Identification of a cis-regulatory element involved inphytochrome down-regulated expression of the pea small GTPase gene pra2,Plant Physiol. 120:491-499(1999); DRE, Arabidopsis thaliana cis-actingelement involved in dehydration, low-temperature, salt stresses,Yamaguchi-Shinozaki K., Shinozaki K., Arabidopsis DNA encoding twodesiccation-responsive rd29 genes, Plant Physiol. 101:1119-1120 (1993);Rushton P. J., Torres J. T., Parniske M., Wernert P., Hahlbrock K.,Somssich I. E., Interaction of elicitor-induced DNA-binding proteinswith elicitor response elements in the promoters of parsley PR1 genes,EMBO J. 15(20):5690-5700 (1996); MSA-like cis-acting element involved incell cycle regulation, Ito M., Criqui M. C., Sakabe M., Ohno T., HataS., Kouchi H., Hashimoto J, Fukuda H., Komamine A., Watanabe A.Cell-cycle regulated transcription of A- and B-type plant cyclin genesin synchronous cultures, Plant J. 11:983-992 (1997), all of which arehereby expressly incorporated by reference in their entireties. Ingeneral, preferred are elements that are not critical to sustaining lifeof the host cell (e.g., not associated with “housekeeping genes” thatare essential for basic cell functions), but are functionally associatedwith regulating transcription of a gene or family of genes that resultin a non-lethal phenotypic change in the plant.

Nic gene product responsive elements can be isolated by screening thepromoter region of genes that are transcriptionally activated by the Nicgene product in the same manner as described herein, or can beidentified by hybridization to SEQ ID NO: 1 herein and subsequentscreening for the ability to bind the Nic gene product in the mannerdescribed below.

Nucleic acid sequences employed in carrying out the present inventioninclude naturally occurring or synthetic fragments with sequencesimilarity to SEQ ID NO:1 or a fragment thereof consisting of,desirably, at least 20-455 consecutive nucleotides, preferably, at least30-400 consecutive nucleotides, more preferably, 50-350 consecutivenucleotides, and, most preferably, 100-300 or 200-400 consecutivenucleotides. This definition is intended to encompass natural allelicvariations of DNA of SEQ ID NO:1 or said fragments. Thus, DNA sequencesthat hybridize to DNA of SEQ ID NO:1, or the complement thereof, mayalso be employed in carrying out the present invention. Preferredembodiments include fragments of SEQ ID NO: 1, or other Nic gene productresponsive elements (i.e., elements that bind to the complement of SEQID NO:1), that retain the ability to bind the Nic gene product. Suchfragments will, in general, be continuous fragments or portions of thenaturally occurring construct that are at least 20, 40 or 60 nucleotidesin length. Conditions which permit other DNA sequences with sequencesimilarity to SEQ ID NO:1 can be determined in a routine manner. Forexample, hybridization of such sequences may be carried out underconditions of reduced stringency or even stringent conditions (e.g.,conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M sodiumcitrate, 0.1% SDS at 60° C. or even 70° C. to DNA with the sequencegiven herein as SEQ ID NO:1 using a standard in situ hybridizationassay. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual(2d Ed. 1989)(Cold Spring Harbor Laboratory)). In general, suchsequences will be at least 65% similar, 75% similar, 80% similar, 85%similar, 90% similar, or even 95% similar, or more, with the sequencegiven herein as SEQ ID NO:1. Determinations of sequence similarity aremade with the two sequences aligned for maximum matching; gaps in eitherof the two sequences being matched are allowed in maximizing matching.Gap lengths of 10 or less are preferred, gap lengths of 5 or less aremore preferred, and gap lengths of 2 or less still more preferred.

The DNA sequence of the present invention may consist essentially of thesequence provided herein (SEQ ID NO:1), or equivalent nucleotidesequences representing alleles or polymorphic variants of these genes,or coding regions thereof.

Use of the phrase “substantial sequence similarity” in the presentspecification and claims means that DNA, RNA or amino acid sequenceswhich have slight and non-consequential sequence variations from theactual sequences disclosed and claimed herein are considered to beequivalent to the sequences of the present invention. In this regard,“slight and non-consequential sequence variations” mean that “similar”sequences (i.e., the sequences that have substantial sequence similaritywith the DNA, RNA, or proteins disclosed and claimed herein) will befunctionally equivalent to the sequences disclosed and claimed in thepresent invention. Functionally equivalent sequences will function insubstantially the same manner to produce substantially the samecompositions as the nucleic acid and amino acid compositions disclosedand claimed herein.

Additional nucleic acid sequence for use with aspects of the inventioninclude the Nic gene product responsive element, which can be obtainedfrom the sequence disclosed in U.S. Pat. No. 5,459,252, herein expresslyincorporated by reference in its entirety. In some embodiments, the Nicgene product responsive element resides between −1000 and −600 or −700bp of the NtQPT1 promoter. Accordingly, some embodiments involve a300-400 nucleotide long fragment of the NtQPT1 promoter that correspondsto the sequence of the NtQPT1 promoter between −1000 and −600 or −700,as disclosed in U.S. Pat. No. 5,459,252.

DNA sequences provided herein can be transformed into a variety of hostcells, as discussed below. A variety of suitable host cells, havingdesirable growth and handling properties, are readily available in theart.

Use of the phrase “isolated” or “substantially pure” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been separated from their in vivo cellular environments through theefforts of human beings. As used herein, a “native DNA sequence” or“natural DNA sequence” means a DNA sequence which can be isolated fromnon-transgenic cells or tissue. Native DNA sequences are those whichhave not been artificially altered, such as by site-directedmutagenesis. Once native DNA sequences are identified, DNA moleculeshaving native DNA sequences may be chemically synthesized or producedusing recombinant DNA procedures as are known in the art. As usedherein, a native plant DNA sequence is that which can be isolated fromnon-transgenic plant cells or tissue. As used herein, a native tobaccoDNA sequence is that which can be isolated from non-transgenic tobaccocells or tissue

2. Nucleic Acid Constructs and Transfer Vectors.

Nucleic acid constructs, or “cassettes,” of the present inventioninclude a cis-acting element such as a Nic gene product responsiveelement as described above, typically as a recombinant construct in alinear or circular nucleic acid that serves as a transfer vector forintroducing the Nic gene product into plant cells.

The construct or cassette may be provided in a DNA construct which alsohas at least one replication system. For convenience, it is common tohave a replication system functional in Escherichia coli, such as ColE1,pSC101, pACYC184, or the like. In this manner, at each stage after eachmanipulation, the resulting construct may be cloned, sequenced, and thecorrectness of the manipulation determined. In addition, or in place ofthe E. coli replication system, a broad host-range replication systemmay be employed, such as the replication systems of the P-1incompatibility plasmids, e.g., pRK290. In addition to the replicationsystem, there will frequently be at least one marker present, which maybe useful in one or more hosts, or different markers for individualhosts. That is, one marker may be employed for selection in aprokaryotic host, while another marker may be employed for selection inan eukaryotic host, particularly the plant host. The markers may beprotection against a biocide, such as antibiotics, toxins, heavy metals,or the like; may provide complementation, by imparting prototrophy to anauxotrophic host; or may provide a visible phenotype through theproduction of a novel compound in the plant.

Nucleic acid constructs of the present invention may include one or morematrix attachment regions positioned 5′, 3′, or both 5′ and 3′ to thecis-acting element(s) to enhance the stability and/or hereditabilitythereof, as described in U.S. Pat. Nos. 5,773,689 to Thompson et al.,U.S. Pat. No. 5,773,695 to Thompson et al., U.S. Pat. No. 6,245,974 toMichalowski et al., U.S. Pat. No. 6,239,328 to Thompson et al., U.S.Pat. No. 6,100,448 to Thompson et al., and U.S. Pat. No. 6,037,525 toThompson et al., the disclosures of which are incorporated by referenceherein in their entirety.

The various fragments comprising the various constructs, cassettes,markers, and the like may be introduced consecutively by restrictionenzyme cleavage of an appropriate replication system, and insertion ofthe particular construct or fragment into the available site. Afterligation and cloning the DNA construct may be isolated for furthermanipulation. All of these techniques are amply exemplified in theliterature as exemplified by J. Sambrook et al., Molecular Cloning, ALaboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

Vectors which may be used to transform plant tissue with nucleic acidconstructs of the present invention include both ballistic vectors andAgrobacterium vectors, as well as vectors suitable for DNA-mediatedtransformation. These are discussed in greater detail below.

The nucleic acid constructs molecules and vectors used to produce thetransformed cells and plants of this invention may further comprise adominant selectable marker gene. Suitable dominant selectable markersfor use in tobacco include, inter alia, antibiotic resistance genesencoding neomycin phosphotransferase (NPTII), hygromycinphosphotransferase (HPT), and chloramphenicol acetyltransferase (CAT).Another well-known dominant selectable marker suitable for use intobacco is a mutant dihydrofolate reductase gene that encodesmethotrexate-resistant dihydrofolate reductase. DNA vectors containingsuitable antibiotic resistance genes, and the corresponding antibiotics,are commercially available.

3. Plant Transformation, Regeneration and Propagation.

Transformed cells are selected out of the surrounding population ofnon-transformed cells by placing the mixed population of cells into aculture medium containing an appropriate concentration of the antibiotic(or other compound normally toxic to the cells) against which the chosendominant selectable marker gene product confers resistance. Thus, onlythose plant cells that have been transformed will survive and multiply.

Methods of making recombinant plants of the present invention, ingeneral, involve first providing a plant cell capable of regeneration(the plant cell typically residing in a tissue capable of regeneration).The plant cell is then transformed with a DNA construct comprising acassette of the present invention (as described herein) and arecombinant plant is regenerated from the transformed plant cell. Asexplained below, the transforming step is carried out by techniques asare known in the art, including but not limited to bombarding the plantcell with microparticles carrying the transcription cassette, infectingthe cell with an Agrobacterium tumefaciens containing a Ti plasmidcarrying the cassette, or any other technique suitable for theproduction of a transgenic plant.

Microparticles carrying a DNA construct of the present invention, whichmicroparticle is suitable for the ballistic transformation of a plantcell, are also useful for making transformed plants of the presentinvention. The microparticle is propelled into a plant cell to produce atransformed plant cell, and a plant is regenerated from the transformedplant cell. Any suitable ballistic cell transformation methodology andapparatus can be used in practicing the present invention. Exemplaryapparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat.No. 4,945,050, and in Christou et al., U.S. Pat. No. 5,015,580 (thedisclosures of all U.S. Patent References cited herein are to beincorporated herein by reference). When using ballistic transformationprocedures, the cassette may be incorporated into a plasmid capable ofreplicating in or integrating into the cell to be transformed. Examplesof microparticles suitable for use in such systems include 1 to 5micrometer (μm) gold spheres. The DNA construct may be deposited on themicroparticle by any suitable technique, such as by precipitation.

Numerous Agrobacterium vector systems useful in carrying out the presentinvention are known. For example, U.S. Pat. No. 4,459,355 discloses amethod for transforming susceptible plants, including dicots, with anAgrobacterium strain containing the Ti plasmid. The transformation ofwoody plants with an Agrobacterium vector is disclosed in U.S. Pat. No.4,795,855. Further, U.S. Pat. No. 4,940,838 to Schilperoort et al.discloses a binary Agrobacterium vector (i.e., one in which theAgrobacterium contains one plasmid having the vir region of a Ti plasmidbut no T region, and a second plasmid having a T region but no virregion) useful in carrying out the present invention.

As a high copy number of decoy sequences must typically be present inthe genome, tandem copies of the cis-acting element(s) could be insertedinto an Agrobacterium vector, but the preferred method of planttransformation is by particle bombardment which introduces multiplecopies of the transgenic DNA into the plant genome. The actual number ofthe cis-acting element (whether each individually present on a vectorsuch as a plasmid, counting multiple copies on a single vector orplasmid, or combinations thereof) that must be inserted into the hostcells (and progeny or daughter cells thereof) to obtain increased ordecreased levels of the protein of interest in the cells and plants ofthe invention will depend in part upon the particular element, but ingeneral will be at least 20, 30 or 50 to about 500, 1,000 or 2,000, ormore.

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art. Fusionof tobacco protoplasts with DNA-containing liposomes or viaelectroporation is known in the art. (Shillito et al., “Direct GeneTransfer to Protoplasts of Dicotyledonous and Monocotyledonous Plants bya Number of Methods, Including Electroporation”, Methods in Enzymology153, 313-36 (1987)).

As used herein, “transformation” refers to the introduction of exogenousDNA into cells, so as to produce transgenic cells stably transformedwith the exogenous DNA. By “stably transformed” is meant that theexogenous nucleic acid is passed to daughter or progeny cells of theinitially transformed cells, and preferably passed to or inherited byprogeny plants of the transformed plants (including sexually andasexually reproduced progeny plants).

Transformed cells are induced to regenerate intact plants throughapplication of cell and tissue culture techniques that are well known inthe art. The method of plant regeneration is chosen so as to becompatible with the method of transformation. After regeneration oftransgenic plants from transformed cells, the introduced DNA sequence isreadily transferred to other plant varieties through conventional plantbreeding practices and without undue experimentation.

For example, to analyze the segregation of the transgenic DNA,regenerated transformed plants (R₀) may be grown to maturity, tested forlevels of the protein of interest, and selfed to produce R₁ plants. Apercentage of R₁ plants carrying the transgenic DNA are homozygous forthe transgenic DNA. To identify homozygous R₁ plants, transgenic R₁plants are grown to maturity and selfed. Homozygous R₁ plants willproduce R₂ progeny where each progeny plant carries the transgenic DNA;progeny of heterozygous R₁ plants will segregate 3:1.

Nicotine serves as a natural pesticide which helps protect tobaccoplants from damage by pests. It may therefore be desirable toadditionally transform low or no nicotine plants produced by the presentmethods with a transgene (such as Bacillus thuringiensis) that willconfer additional insect protection.

A preferred plant for use in the present invention is any species of thegenus Nicotiana, or tobacco, including N tabacum, N rustica and Nglutinosa. Any strain or variety of tobacco may be used.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, callus tissue, existing meristematictissue (e.g., apical meristems, axillary buds, and root meristems), andinduced meristem tissue (e.g., cotyledon meristem and hypocotylmeristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the cassette); the plants may comprise grafts of transformed anduntransformed tissues (e.g., a transformed root stock grafted to anuntransformed scion in citrus species). The transformed plants may bepropagated by a variety of means, such as by clonal propagation orclassical breeding techniques. For example, first generation (or T1)transformed plants may be selfed to give homozygous second generation(or T2) transformed plants, and the T2 plants further propagated throughclassical breeding techniques. A dominant selectable marker (such asnpt11) can be associated with the construct to assist in breeding.

In some preferred embodiments of the invention, to help insure that asufficient number of decoy or cis-acting elements are inserted in cellsand retained over many cell divisions to produce a transgenic plant withaltered levels of protein or proteins therein, biolistic transformationis used as described above, circular DNA or plasmids are used to carrythe cis-acting decoy segments as described above, the circular DNA orplasmids that are used are relatively small (e.g., they consist of lessthan 10,000 or less than 6,000 base pairs), and a high molar ratio ofthe cis-acting element to selectable marker (e.g., 10 to 1) is insertedinto the host cells.

As used herein, a crop comprises a plurality of plants of the presentinvention, and of the same genus, planted together in an agriculturalfield. By “agricultural field” is meant a common plot of soil or agreenhouse. Thus, the present invention provides a method of producing acrop of plants having altered levels of a protein of interest, (e.g.,QPTase and PMTase activity and thus having decreased nicotine levels),compared to a similar crop of non-transformed plants of the same speciesand variety.

While the invention describes methods to reduce nicotine levels intransgenic tobacco, this method can also be employed to phenocopymutations in trans-acting transcriptional activators and repressorswithout cloning their respective genomic loci. Promoter regions of agene can be analyzed, using technology known by those skilled in theart, to define regions of the promoter that respond to transcriptionfactors. Typically, this is done by deletion analysis of the promoter.Nested deletions of the promoter are fused to a reporter gene andexpression of the reporter gene is monitored in transgenic organisms.Isolation of transcription factors using current technologies is verydifficult; the present invention circumvents the necessity of cloningthe cognate transcription factors for applications in which it isdesirable to disrupt any set of genes that are coordinately regulated byone or more transcriptional activators. Conversely, the process wouldup-regulate the expression of any set of genes that are coordinatelyregulated by one or more transcriptional repressor.

As noted above, the present invention could be employed to disrupt geneexpression and down-regulate the expression of a protein of interestthat is under the control of a cis-acting activating element in avariety of host cells, including plant (particularly vascular plant suchas monocot and dicot), animal (avian, mammalian), fungi, or bacteriacells, both in vivo and in vitro. In bacteria and fungi, multicopyplasmids can be used to increase copies of molecular decoy present inthe cell.

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Localization of cis-Acting Element in NtQPT1 Promoter

To characterize the minimal sequence required for the NtQPT1 cis-actingelement, the promoter region of the NtQPT1 gene was isolated, truncatedat the 5′ end, and fused to the gene encoding β-glucuronidase (GUS) toassess function as a specific enhancer of nicotine production. TheNtQPT1 gene was isolated and sequenced. The start of the transcript wasdetermined by comparing the TobRD2 cDNA sequence to the genomic locussequence. Sequence located 5′ of the transcription start site wasdefined as promoter sequence. Using PCR primers and the promoter as atemplate, truncations were made at the 5′ end of the promoter todetermine minimal cis-acting enhancer sequence (see FIG. 2). Thetruncations were fused to the uidA gene, which encodes GUS. The fusiongene was inserted into a vector and transformed by standard methods ofballistic transformation into Nicotiana tabacum Burley 21.

GUS activity was assessed by dividing plants into roots, stems, andleaves. Each plant tissue, transformed with a different NtQPT1truncation construct, was ground with a mortar and pestle, proteins wereextracted with NaPO₄ buffer, pH 7.0, X-Glc (100 ug/mL) was added, andthe assay was carried out at 37° C. for 30 min. GUS activity wasmeasured at 595 nm). For each construct at least 20 independenttransformants were assayed. A mean and standard deviation wasdetermined. GUS activity in truncations was compared to a CaMV 35S-GUSfusion and a promoterless GUS (pBI101) control. Maximal GUS activity,representing NtQPT1 expression, was obtained when −586 to −2000 bp wasfused to the uidA gene (FIG. 3). Shorter promoters from −1 to −586 didnot support high levels of uidA expression. Therefore, the NtQPT1cis-acting element is located between −586 and −2000 bp 5′ of thetranscription start site.

EXAMPLE 2 Localization of Nic Gene Product Binding Site in NtQPT1Promoter

The NtQPT1 promoter deletion series fused to the uidA reporter gene(encoding GUS) was transformed into nic⁻/nic⁻ homozygous N. tabacumplants. R₀ transformants having the transgenic DNA at a single locuswere crossed with nic⁻/nic⁻ or Nic⁺/Nic⁺ homozygous plants to givehomozygous (nic⁻/nic⁻) and heterozygous (Nic⁺/nic⁻) progeny carrying thetransgene (NtQPT1 promoter-GUS) at the same chromosomal location. GUSactivity was quantified in multiple progeny from multiple, independenttransformants and compared between Nic⁺ and nic⁻ phenotypes (TABLE 1).Ratios greater than 1.5 were determined to contain the cis-actingelements responding to Nic gene product activation. TABLE 1 Regulationof NtQPT1 Promoter-Directed GUS Expression by the Nic Gene Products inTobacco. GUS Independent activity in GUS activity in GUS ratio PromoterTransformants Nic⁺/nic⁻² nic⁻/nic⁻² of Nic/nic 2.0 (2010)¹ 2 111.7 (7) 21.2 (5) 5.3 46.6 (6) 11.9 (8) 5.6 1.3 (1306)¹ 2 92.4 (6) 16.6 (4) 5.593.9 (7) 12.9 (5) 7.3 1.0 (1042)¹ 3 55.7 (6) 18.3 (4) 3.0 74.1 (6) 27.5(7) 2.7 78.0 (5) 17.2 (7) 4.5 734 1  5.5 (5)  3.5 (5) 1.5 586 3 47.55(5)  44.9 (5) 1.06 24.3 (3)  16.0 (36) 1.5  29.1 (33)  30.3 (19) 0.97535 3  71.6 (10) 50.3 (5) 1.4 54.0 (5) 40.7 (3) 1.3 51.9 (5) 67.8 (5)0.8 CaMV 35S 4 32.7 (4) 19.6 (4) 1.7 44.8 (6) 47.6 (3) 0.94 54.8 (5)40.6 (5) 1.3  9.7 (4)  8.6 (3) 1.1GUS activity is expressed as pmol MU/μg protein/min.¹Actual promoter size (bp) is indicated in parenthesis.²Number in parenthesis indicates the number of plants tested.

These experiments demonstrated that binding of Nic gene products islocated between approximately −1000 and −600 or −700 bp of the NtQPT1promoter as determined by GUS activity in Nic⁺/nic⁻ and nic⁻/nic⁻plants.

EXAMPLE 3 Regulation of NtQPT1 Gene Expression Using Molecular Decoys

Nucleotide sequence located between −1000 and −600 or −700 bp of theNtQPT1 promoter is inserted in tandem arrays into a plant-Agrobacteriumshuttle vector and subsequently transformed into tobacco via methodsknown to one skilled in the art. Plants stably transformed with saidvector are assessed for the level of expression of NtQPT1 and fornicotine and/or TSNA content. These experiments will demonstrate thattobacco transformed with molecular decoys that interact with Nic geneproducts will exhibit a reduced amount of nicotine and/or TSNA. Plantswith multiple tandem insertions of the molecular decoy which havereduced NtQPT1 expression and reduced nicotine levels are used forexpression of commercially valuable products and production of tobaccoproducts having reduced nicotine and/or TSNA content.

EXAMPLE 4 Regulation of ASF-1 Binding Using a TGACG Molecular Decoy

The nucleotide sequence TGACG is inserted in tandem arrays into aplant-Agrobacterium shuttle vector and transformed into a plant such aspea via methods known to one skilled in the art. Plants stablytransformed with said vector have reduced binding activity oftrans-acting DNA binding factor ASF-1 which recognizes the sequencemotif TGACG which is found in plant genes such as histone genes (Mikamiet al., (1987) FEBS Lett. 223:273); enzyme genes for agropinebiosynthesis (Velten et al., EMBO J. 3:2723-30); the octopine synthasegene (Ellis et al., EMBO J. 6:3203); and the mannopine synthase gene(DeRita and Gelvin, (1987) Mol. Gen. Genet. 207:233); as well as theCaMV35S gene, histone H3 gene and nopaline synthase gene.

EXAMPLE 5 Regulation of Spatial and Temporal Expression ofBeta-Phaseolin Using Molecular Decoys

The nucleotide sequence corresponding to UAS1 (−295 to −109) of thebeta-phaseolin gene is inserted in tandem arrays into aplant-Agrobacterium shuttle vector and transformed into a bean plant viamethods known to one skilled in the art. Plants stably transformed withsaid vector have reduced binding activity of trans-acting DNA bindingfactor PvALF which recognizes the sequences CATGCAAA and CATGCATGlocated in UAS1 (Bobb et al. (1997) Nucleic Acids Res 25(3):641-7).Plants with reduced binding of PvALF would have reduced expression ofseed-specific expression of beta-phaseolin primarily in cotyledons andshoot meristem (Bustos et al. (1991) EMBO J. 10(6):1469-1479).

Transformation of tandem arrays of nucleotide sequences corresponding tothe vicilin-box (GCCACCTCAA; SEQ ID NO:2) and site B (CACACGTCAA; SEQ IDNO:3) of the beta-phaseolin gene into a bean plant results in thereduced binding activity of trans-acting DNA binding factors ROM 1 andROM2 leading to premature onset of beta-phaseolin expression. ROM1 andROM2 proteins function as repressors of beta-phaseolin andphytohemagglutinin L-subunit expression to block onset of seedmaturation (U.S. Pat. No. 6,160,202 to Bustos; Chern et al. (1996) PlantCell 8:305-321; Chern et al. (1996) Plant J. 10:135-148).

EXAMPLE 6 Regulation of Plant Gene Expression Using Molecular Decoys

Transformation of tobacco plants with tandem arrays of the root-specificcis-acting element from the tobacco RB7 promoter (U.S. Pat. No.5,459,252 to Conkling et al.; Yamamoto et al. (1991) Plant Cell12:3399-3406), which codes for a structural gene, results in the reducedbinding activity of the trans-acting DNA binding factor of the RB7cis-acting element.

Likewise, similar tandem arrays of the following cis-elements aretransformed into the plants to reduce binding activity of thecorresponding trans-acting DNA binding factors: the AATT cis-actingrepeat element and its corresponding PABF trans-acting factor (see U.S.Pat. Nos. 5,834,236 and 6,191,258); the positive poly(dA-dT) regulatoryelement and binding protein and negative CCAA repeat element and bindingprotein (Wang et al. (1992) Mol. Cell Biol. 12:3399-3406); the root-tipregulatory element from the tobacco phytochrome A1 promoter of tobacco(Adam et al. (1995) Plant Mol. Biol. 29:983-993); theanaerobiosis-responsive element from the maizeglyceraldehyde-3-phosphate dehydrogenase 4 gene (Geffers et al. (2000)Plant Mol. Biol. 43:11-2 1); and the seed-specific regulatory regionfrom an Arabidopsis oleosin gene (see U.S. Pat. No. 5,792,922).

EXAMPLE 7 Tobacco Having Reduced Nicotine and/or TSNA Levels GeneratedUsing Molecular Decoys

Multiple copies of an approximately 300 or 400 nucleotide long fragmentof the NtQPT1 promoter (e.g., including nucleotide sequence locatedbetween −1000 and −600 or −700 bp of the NtQPT1 promoter, such as SEQ IDNO:1) are affixed to microparticles (e.g., by precipitation) that aresuitable for the ballistic transformation of a plant cell (e.g., 1 to 5μm gold spheres). The microparticles are propelled into tobacco plantcells (e.g., Burley 21 LA) so as to produce transformed plant cells, andplants are regenerated from the transformed plant cells. Burley 21 LA isa variety of Burley 21 with substantially reduced levels of nicotine ascompared with Burley 21 (i.e., Burley 21 LA has 8% the nicotine levelsof Burley 21, see Legg et al., Can J Genet Cytol, 13:287-91 (1971); Legget al., J Hered, 60:213-17 (1969)) Any suitable ballistic celltransformation methodology and apparatus can be used. Exemplaryapparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat.No. 4,945,050, and in Christou et al., U.S. Pat. No. 5,015,580, both ofwhich are herein expressly incorporated by reference in theirentireties. Optionally, the transformed nucleic acid can include a geneencoding a selectable marker (e.g., a marker that allows for positive ornegative selection of transformants) or the molecular decoys can beco-transferred with a selectable marker gene. In this manner, positivetransformants can be easily identified.

Transformed cells, tissues, and seedlings are grown on Murashige-Skoog(MS) medium (with or without the selection compound, e.g., antibiotic,depending on whether a selectable marker was used. One-hundredindependent transformants of Burley 21 LA (T₀) are allowed to self.Progeny of the selfed plants (T₁) are germinated. Nicotine levels of T₁progeny are measured qualitatively using a micro-assay technique.Approximately ˜200 mg fresh tobacco leaves are collected and ground in 1ml extraction solution. (Extraction solution: 1 ml Acetic acid in 100 mlH₂O) Homogenate is centrifuged for 5 min at 14,000×g and supernatantremoved to a clean tube, to which the following reagents are added: 100μL NH₄OAC (5 g/100 ml H₂O+50 μL Brij 35); 500 μL Cyanogen Bromide (SigmaC-6388, 0.5 g/100 ml H₂O +50 μBrij 35); 400 μL Aniline (0.3 ml bufferedAniline in 100 ml NH₄OAC+50 μL Brij 35). A nicotine standard stocksolution of 10 mg/ml in extraction solution is prepared and diluted tocreate a standard series for calibration. Absorbance at 460 nm is readand nicotine content of test samples are determined using the standardcalibration curve.

T₁ progeny that have less than 10% of the nicotine levels of the Burley21 LA parent are allowed to self to produce T₂ progeny. Homozygous T₂progeny are then identified. Nicotine levels in homozygous andheterozygous T₂ progeny are also qualitatively determined using themicro-assay. Leaf samples of homozygous T₂ progeny can also be sent tothe Southern Research and Testing Laboratory in Wilson, N.C. forquantitative analysis of nicotine levels using Gas Chromatography/FlameIonization Detection (GC/FID). Homozygous T₂ progeny of will havenicotine levels that are substantially reduced as compared to theuntransformed tobacco (e.g., ˜70 ppm). Because the nicotine levels insuch plants are substantially reduced, the TSNA levels in these plantsis concomitantly reduced.

These experiments will demonstrate that tobacco transformed withmolecular decoys that interact with Nic gene products will exhibit areduced amount of nicotine and/or TSNA. Plants with multiple tandeminsertions of the molecular decoy which have reduced NtQPT1 expressionand reduced nicotine levels are used for expression of commerciallyvaluable products and production of tobacco products having reducednicotine and/or TSNA content.

EXAMPLE 8 Low Nicotine and TSNA blended Tobacco

The following example describes several ways to create tobacco productshaving specific amounts of nicotine and/or TSNAs through blending. Someblending approaches begin with tobacco prepared from varieties that haveextremely low amounts of nicotine and/or TSNAs. By blending preparedtobacco from a low nicotine/TSNA variety (e.g., undetectable levels ofnicotine and/or TSNAs ) with a conventional tobacco (e.g., Burley, whichhas 30,000 parts per million (ppm) nicotine and 8,000 parts per billion(ppb) TSNA; Flue-Cured, which has 20,000 ppm nicotine and 300 ppb TSNA;and Oriental, which has 10,000 ppm nicotine and 100 ppb TSNA), tobaccoproducts having virtually any desired amount of nicotine and/or TSNAscan be manufactured. Tobacco products having various amounts of nicotineand/or TSNAs can be incorporated into tobacco use cessation kits andprograms to help tobacco users reduce or eliminate their dependence onnicotine and reduce the carcinogenic potential.

For example, a step 1 tobacco product can be comprised of approximately25% low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2tobacco product can be comprised of approximately 50% low nicotine/TSNAtobacco and 50% conventional tobacco; a step 3 tobacco product can becomprised of approximately 75% low nicotine/TSNA tobacco and 25%conventional tobacco; and a step 4 tobacco product can be comprised ofapproximately 100% low nicotine/TSNA tobacco and 0% conventionaltobacco. A tobacco use cessation kit can comprise an amount of tobaccoproduct from each of the aforementioned blends to satisfy a consumer fora single month program. That is, if the consumer is a one pack a daysmoker, for example, a single month kit would provide 7 packs from eachstep, a total of 28 packs of cigarettes. Each tobacco use cessation kitwould include a set of instructions that specifically guide the consumerthrough the step-by-step process. Of course, tobacco products havingspecific amounts of nicotine and/or TSNAs would be made available inconveniently sized amounts (e.g., boxes of cigars, packs of cigarettes,tins of snuff, and pouches or twists of chew) so that consumers couldselect the amount of nicotine and/or TSNA they individually desire.There are many ways to obtain various low nicotine/low TSNA tobaccoblends using the teachings described herein and the following isintended merely to guide one of skill in the art to one possibleapproach.

To obtain a step 1 tobacco product, which is a 25% low nicotine/TSNAblend, prepared tobacco from an approximately 0 ppm nicotine/TSNAtobacco can be mixed with conventional Burley, Flue-cured, or Orientalin a 25%/75% ratio respectively to obtain a Burly tobacco product having22,500 ppm nicotine and 6,000 ppb TSNA, a Flue-cured product having15,000 ppm nicotine and 225 ppb TSNA, and an Oriental product having7,500 ppm nicotine and 75 ppb TSNA. Similarly, to obtain a step 2product, which is 50% low nicotine/TSNA blend, prepared tobacco from anapproximately 0 ppm nicotine/TSNA tobacco can be mixed with conventionalBurley, Flue-cured, or Oriental in a 50%/50% ratio respectively toobtain a Burly tobacco product having 15,000 ppm nicotine and 4,000 ppbTSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb TSNA,and an Oriental product having 5000 ppm nicotine and 50 ppb TSNA.Further, a step 3 product, which is a 75%/25% low nicotine/TSNA blend,prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco canbe mixed with conventional Burley, Flue-cured, or Oriental in a 75%/25%ratio respectively to obtain a Burly tobacco product having 7,500 ppmnicotine and 2,000 ppb TSNA, a Flue-cured product having 5,000 ppmnicotine and 75 ppb TSNA, and an Oriental product having 2,500 ppmnicotine and 25 ppb TSNA.

It should be appreciated that tobacco products are often a blend of manydifferent types of tobaccos, which were grown in many different parts ofthe world under various growing conditions. As a result, the amount ofnicotine and TSNAs will differ from crop to crop. Nevertheless, by usingconventional techniques one can easily determine an average amount ofnicotine and TSNA per crop used to create a desired blend. By adjustingthe amount of each type of tobacco that makes up the blend one of skillcan balance the amount of nicotine and/or TSNA with other considerationssuch as appearance, flavor, and smokability. In this manner, a varietyof types of tobacco products having varying level of nicotine and/ornitrosamine, as well as, appearance, flavor and smokeability can becreated.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allreferences cited herein are hereby expressly incorporated by reference.

1-73. (canceled)
 74. A transgenic tobacco plant having a reduced amountof nicotine therein, wherein said transgenic tobacco plant comprisescells comprising an exogenous nucleic acid sequence that binds a Nicgene product, wherein said exogenous nucleic acid sequence is selectedfrom the group consisting of: (a) an isolated nucleic acid sequence ofSEQ ID NO:1 or an active fragment thereof, wherein said fragmentcomprises 20 to 455 consecutive nucleotides of SEQ ID NO:1 and binds aNic gene product; (b) an isolated nucleic acid sequence which is atleast 80% identical to the nucleic acid sequence of (a) and binds a Nicgene product; and (c) an isolated nucleic acid that hybridizes understringent conditions to the complement of the nucleic acid sequence of(a) and binds a Nic gene product, and wherein said exogenous nucleicacid is present in said cells in a copy number sufficient to reduce theamount of nicotine in said tobacco plant as compared to the amount ofnicotine that would be present in said plant in the absence of saidexogenous nucleic acid sequence, and wherein the transgenic tobaccoplant further comprises a transgene encoding a heterologous protein ofinterest.
 75. The transgenic tobacco plant of claim 1, wherein saidexogenous nucleic acid sequence consists essentially of the nucleotidesequence of SEQ ID NO:1.
 76. The transgenic tobacco plant of claim 1,wherein said exogenous nucleic acid sequence is contained within arecombinant nucleic acid construct, wherein said recombinant nucleicacid construct does not contain a NtQPT1 coding sequence.
 77. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment comprising 30-400 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 78. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment comprising 50-350 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 79. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment comprising 100-300 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 80. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment comprising 200-400 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 81. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment of at least 20 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 82. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment of at least 40 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 83. Thetransgenic tobacco plant of claim 1, wherein said isolated nucleic acidsequence of SEQ ID NO:1 is a fragment of at least 60 consecutivenucleotides of SEQ ID NO:1 and binds a Nic gene product.
 84. A method ofproducing a heterologous protein of interest in a plant, comprising: a)growing the transgenic tobacco plant of claim 1; and b) collecting theheterologous protein of interest from said transgenic plant.
 85. Amethod of producing a heterologous protein of interest in a plant,comprising: a) growing the transgenic tobacco plant of claim 2; and b)collecting the heterologous protein of interest from said transgenicplant.
 86. A method of producing a heterologous protein of interest in aplant, comprising: a) growing the transgenic tobacco plant of claim 3;and b) collecting the heterologous protein of interest from saidtransgenic plant.
 87. A method of producing a heterologous protein ofinterest in a plant, comprising: a) growing the transgenic tobacco plantof claim 9; and b) collecting the heterologous protein of interest fromsaid transgenic plant.
 88. A method of producing a heterologous proteinof interest in a plant, comprising: a) growing the transgenic tobaccoplant of claim 10; and b) collecting the heterologous protein ofinterest from said transgenic plant.
 89. A method of producing aheterologous protein of interest in a plant, comprising: a) growing thetransgenic tobacco plant of claim 11; and b) collecting the heterologousprotein of interest from said transgenic plant.
 90. The transgenictobacco plant of claim 1, wherein the heterologous protein of interestis a Bacillus thuringiensis insecticidal protein.
 91. The method ofclaim 12, wherein the heterologous protein of interest is a Bacillusthuringiensis insecticidal protein.